This project aims to understand the non-canonical activity of retinoic acid (RA) signaling. These studies will employ embryonic stem (ES) cell culture as the experimental model system. Oct4 (Pou5f1) is the master regulator of pluriopotent stem cells including ES, and its expression level must be maintained within a narrow range to ensure the stemness feature of ES. A constant level of Oct4 expression depends upon promyelocytic leukemia (Pml) protein and a particular nuclear structure called Pml-nuclear body (Pml-NB). Our preliminary data show RA exerts a non-canonical activity that rapidly activates Extracellular signal Regulated Kinase 1/2 (ERK1/2), regulates specific proteins' post-translational modification (PTM) and disrupts Pml-NB with which the Oct4 basal promoter is physically associated. This Pml-NB structure is also important for inter- chromosomal coordination of the Oct4 locus with another key stem cell locus Nanog located on a different chromosome. Therefore, the non-canonical activity of RA disrupts Pml-NB, lowers Oct4 and Nanog expression levels and affects the stemness property of ES. We propose a principal hypothesis that in ES cells, the non- canonical RA-activated ERK1/2 alters specific proteins' PTM and regulates the Pml-NB structure which is critical to coordinated regulation of multiple key genomic loci such as Oct4 and Nanog, and ultimately affects the stem cell fate. We propose two aims to understand these newly identified effects of RA. Specifically, aim 1 will examine the non-canonical activity of RA transmitted, at least partially, through ERK1/2 activation to post- translationally modify specific proteins. Aim 2 will examine how this activity rapidly affects the sub-nuclear structure Pml-NB to facilitate an environment coordinating multiple gene regulation/chromatin remodeling of multiple loci such as Oct4/Nanog loci in ES cells, thereby affecting stem cell fate. Completion of these studies will allow us to construct a more comprehensive picture of how RA signal can be transmitted, through canonical and non-canonical pathways, to modulate the overall health of the cells including ES cells. Understanding these new mechanisms of action of RA will facilitate the manipulation of these specific RA signaling pathways to improve the efficacy of its application as a therapeutic agent/nutrient, and to minimize its serious toxic effects. These studies will also shed lights on the maintenance of ES cell culture with respect to its extreme sensitivity to RA/vitamin A in the culture medium. Furthermore, this non-canonical RA activity may be a key to epigenetic changes caused by RA applied as a therapeutic agent in diseased conditions.